The present invention relates to apparatus and methods for processing or separating compounds, particularly biomolecules in the form of recombinant proteins produced by recombinant microorganisms and the removal of small molecular mass macromolecules from solutions.
Processing solutions of biomolecules often involves the use of various buffer solutions. During commercial processing of biomolecules for example, large volumes of buffers are required which can be costly and also may cause problems for disposal. Often, spent buffers contain macromolecular waste materials which not only prevent further use of the buffers but also need to be disposed of safely.
In the past, a preparative electrophoresis technology for macromolecule separation which utilises tangential flow across a polyacrylamide membrane when a charge is applied across the membrane was used to separate macromolecules. The general design of the earlier system facilitated the purification of proteins and other macromolecules under near native conditions. The technology is bundled into a cartridge comprising several membranes housed in a system of specially engineered grids and gaskets which allow separation of macromolecules by charge and/or molecular weight. The system can also concentrate and desalt/dialyse at the same time. The multi-modal nature of the system allows this technology to be used in a number of other areas especially in the production of biological components for medical use.
The effect of low molecular weight proteins and ions building up in the buffer stream can slow the transfer of proteins during purification. These contaminants carry current which lessens target protein migration and can lead to heat build up in the protein solution. There is a need to be able to remove contaminants from the buffers streams in such electrophoretic systems.
It has been discovered that the buffer stream can be processed to remove these low molecular weight contaminants. By cycling the buffer stream through a separate apparatus, it is possible to remove the majority of the proteins/contaminants present in the buffer stream whilst maintaining the conductivity and pH of the buffer.
The modern biotechnology industry is faced with a number of problems especially concerning the processing of biomolecules produced recombinantly. Expression of recombinant genes in recombinant cells is often low and purification from the host cell difficult. The starting sample is usually dilute and the need to concentrate the sample by conventional means can give low recoveries. Usually, a flag peptide attached to the recombinant protein is used (often a six histidine peptide is added to the protein) to enable purification of the protein. This tag can interfere with the biological function by effecting folding for example, leading to inactive protein and incorrect assessment of the construct.
Presently, the purification of biomolecules, particularly recombinant proteins is sometimes a long and cumbersome process especially when purifying recombinant biomolecules.
It is desirable to have a preparative electrophoretic system which can efficiently and effectively separate macromolecules, such as recombinant proteins, and can remove contaminants from product samples and buffer streams.
In accordance with the present invention, there is provided an electrophoretic system which efficiently and effectively separates macromolecules and can remove contaminants from samples and buffer streams.
Further, in accordance with the present invention, there is provided an apparatus for separating macromolecules by electrophoretic separation, the apparatus comprising:
(a) a first cathode in a first cathode zone;
(b) a first anode in a first anode zone, the anode disposed relative to the first cathode so as to be adapted to generate a first electric field in a first electric field area therebetween upon application of a first voltage potential between the first cathode and the first anode;
(c) a first electrophoretic buffer disposed in the first cathode zone and the first anode zone;
(d) a first separation membrane having a defined molecular mass cut-off disposed in the first electric field area;
(e) a first restriction membrane disposed between the first cathode zone and the first separation membrane so as to define a first interstitial volume therebetween;
(f) a second restriction membrane disposed between the first anode zone and the first separation membrane so as to define a second interstitial volume therebetween;
(g) means adapted to provide a sample constituent in a selected one of the first interstitial and second interstitial volumes wherein upon application of the first voltage potential, a selected separation product is removed from the sample constituent through the first separation membrane and provided to the other of the first and second interstitial volumes;
(h) a second cathode zone optionally containing a second anode;
(i) a second anode zone optionally containing a second anode, the second anode zone disposed relative to the second cathode zone so as to be adapted to generate a second electric field area therebetween upon application of an optional second voltage potential between the optional second cathode and the optional second anode;
(j) a second electrophoretic buffer disposed in the second anode zone and the second cathode zone;
(k) a second separation membrane having a defined molecular mass cut-off disposed in the second electric field area;
(l) a third restriction membrane disposed between the second anode zone and the second separation membrane so as to define a third interstitial volume therebetween;
(m) a fourth restriction membrane disposed between the second cathode zone and the second separation membrane so as to define a fourth interstitial volume therebetween;
(n) means adapted to provide the first electrophoretic buffer to a selected one of the third and fourth interstitial volumes wherein a selected separation product is removed from the first electrophoretic buffer through the second separation membrane, and provided to the other of the third and fourth interstitial volumes while substantially preventing the first electrophoretic buffer from entering the other of the third and fourth interstitial volumes; and
(o) means adapted to provide the first electrophoretic buffer after the selected separation product has been removed from the first electrophoretic buffer to a selected one of the first cathode and anode zones.
Still further, in accordance with the present invention, there is provided in a first general aspect, a system for separating macromolecules by electrophoresis, the system comprising:
(a) a first cathode compartment and a first anode compartment;
(b) a first cathode and first anode positioned in the respective compartments;
(c) a first electrophoresis buffer stream feeding the first cathode and first anode compartments;
(d) a first chamber and a second chamber positioned on either side of a first ion-permeable separation membrane having a defined molecular mass cut-off, the first chamber and the second chamber being positioned between the cathode and the anode compartments and separated by an ion-permeable restriction membrane positioned on each side of the separation membrane, the restriction membrane allowing flow of ions into and out of the compartments and chambers under the influence of an electric field but substantially restrict movement of at least one macromolecule type from the second chamber into a compartment;
(e) a second cathode compartment and a second anode compartment;
(f) optionally, a second cathode and second anode positioned in the respective second cathode and anode compartments;
(g) a second electrophoresis buffer stream feeding the second cathode and second anode compartments;
(h) a third chamber and a fourth chamber positioned on either side of a second ion-permeable separation membrane having a defined molecular mass cut-off, the third chamber and the fourth chamber being positioned between the second cathode and the second anode compartments and separated by third and fourth ion-permeable restriction membranes positioned on each side of the second separation membrane.
An advantage of the present invention is that the electrophoretic system which efficiently and effectively separates macromolecules and can remove contaminants from samples and buffer streams.
Another advantage of the present invention is that recombinant molecules do not require a flag peptide (or peptide tag in general) for its purification. Removing the requirement of a flag/tag on recombinant molecules eliminates the potential of the flag/tag region to interfere with biological function of the molecule. Therefore flag/tag sequences are not required in the vector of the cloned recombinant molecule.
Another advantage is that the electrophoretic separation method of the present invention can result in yields of greater than about 70% with purity at least about 90%.
Still another advantage is that the electrophoretic separation method of the present invention is able to be scaled-up without denaturing or adversely altering the physical or biological properties of recombinant proteins separated by the present invention.
These and other advantages and benefits of the invention will be apparent to those skilled in the art upon reading and understanding of the following detailed description.